1. Field of the Invention
The present invention generally relates to test materials and more particularly to a process for passively adsorbing immuno-reactive haptens to solid phases to permit immunoassays of increased sensitivity.
2. Prior Art
Haptens are classically measured by the competitive inhibition assay first described by Yalow and Berson (J. Clin. Invest. 39: 1157, 1960). This assay is a radioimmunoassay carried out by mixing a limiting amount of hapten-specific antibody with specified volumes of a sample containing an unknown amount of a hapten and a solution containing a known amount of the same hapten or an analog of the same hapten. Unlabeled and labeled haptens then compete for a limited number of antibody-binding sites. By separating the free and the antibody-bound labeled hapten into distinct fractions or phases and then measuring the amount of radioactivity in each of these two phases, one can quantitatively determine the amount of hapten in the sample being tested. Phase separation of the free hapten and the antibody-bound hapten can be accomplished by several methods which are currently in practice, including the use of species-specific antiglobulin to precipitate hapten-antibody complexes. Charcoal, ion exchange resins and various other types of solid phases have also been used to bind specific antibody.
The general methodologies and principles which are utilized in the radioisotope immunoassay methods employing phase separation to quantitate haptens have more recently also been applied to systems which employ reagents which are labeled with enzymes instead of radioisotopes. One such system is known as the enzyme-linked immunosorbent assay (ELISA), utilizing enzyme-hapten conjugates and a plastic solid phase to which hapten-specific antibody is adsorbed to effect phase separation. As in the case of radioimmunoassays, sample hapten and enzyme-labeled hapten compete for a limited number of antibody-combining sites on the solid phase. The amount of antibody-bound labeled hapten (which has an inverse relationship to the amount of hapten in the unknown sample) is determined by measuring the enzymatic activity of the solid phase.
A second system known as the enzyme multiplication immunoassay technique (EMIT) is similar to ELISA and certain radioimmunoassays in that enzyme-labeled hapten and sample hapten compete for a limited number of antibody-binding sites. However, EMIT does not require phase separation because the enzyme-hapten conjugate is prepared in such a manner that the enzyme will not react with substrate when antibody is bound to the enzyme-hapten conjugate. Consequently, EMIT specifically measures the amount of free or unbound enzyme-hapten conjugate.
There are few examples of hapten assays in which hapten itself is bound to a solid phase. One such example is the competetive enzyme-linked immunoassay (CELIA) described by Yorde et al. (Clin. Chem. 22:1372, 1976). In the CELIA system free hapten in an unknown sample and hapten covalently bound to a solid phase, such as Sepharose beads which are cross-linked dextran, compete for a limiting number of antibody (1st antibody) binding sites in solution. The quantity of hapten in the unknown sample is determined by measuring the amount of specific antibody bound to the solid phase. This measurement is accomplished by an enzymatic technique in which anti-globulin bound to 1st antibody and anti-enzyme-enzyme immune complex are added in sequence. The anti-globulin functions to bridge the bound hapten specific antibody with the anti-enzyme-enzyme immune complex which is the indicator system. One then measures the enzymatic activity of the solid phase in the presence of substrate.
A radioimmunoassay, similar to CELIA in principle, has been developed by Zollinger and Mandrell (Infect. and Immun. 18:424, 1977) to serotype bacteria. In this procedure, specific typing antibody is preincubated with a heterologous antigen (distinct form hapten) preparation. The reaction mixture is then added to microtiter wells passively coated with antigen homologous to the typing antisera. The amount of antibody bound to the solid phase (which has an inverse relationship with the degree of antigen similarity shared by the heterologous test antigen and the solid phase homologous antigen) is determined by adding a radio-labeled anti-globulin and then measuring the amount of label bound to the solid phase.
The primary advantage of using a solid-phase hapten, that is a hapten bound to a solid phase, as in the CELIA procedure, is that the sensitivity of the hapten assay can be increased dramatically by using labeled anti-globulin as the indicator system rather than labeled hapten.
The reason for enhanced sensitivity pertains to the increased number of enzyme labels that can be linked to antibody relative to hapten molecules. Thus, three to four enzyme molecules can be successfully linked to a single antibody, resulting in a conjugate that will demonstrate both antibody and enzymatic activity. In contrast, a single hapten molecule can be labeled with only one enzyme molecule. In reality, the final product is a population of enzyme molecules to which three or four hapten molecules are covalently bound.
If a solid phase hapten were employed in a competitive immunoassay, each hapten specific antibody reacting with the solid phase would result in either three to four to six to eight enzyme labels being bound, depending on whether labeled hapten specific or labeled second antibody were employed. In contrast, if one employed a hapten specific antibody solid phase in a competitive immunoassay, each solid phase antibody could react with two labeled hapten molecules resulting in only two enzyme labels being bound. Thus, the enhanced sensitivity of the immunoassays which employ solid phase hapten and labeled antibody is due to increased amplification of the serologic reaction by virtue of having more labels involved on a unit basis. Unfortunately, simple inexpensive methods of attaching haptens to solid phases have not heretofore existed. It therefore would be desirable to provide a simple, inexpensive, rapid, effective and reproducible method for attaching haptens to solid phases in order to improve the sensitivity of a hapten immunoassay,